Laser drilling technique for creating nanoscale holes

ABSTRACT

A method of forming extremely small pores in a substrate that is used, for example, in patch clamp applications is provided that employs an energy absorbing material beyond a back side of the substrate to allow a laser to be focused adjacent the exit side of the substrate so as to generate a pore through the substrate and can also form a crater in the back side of the substrate and in which the pore may propagate from the crater in a drilling direction that can oppose a laser transmission direction.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

N/A.

CROSS REFERENCE TO RELATED APPLICATION

N/A.

BACKGROUND OF THE INVENTION

The present invention relates to electrophysiology and “patch clamping”for investigating ionic and molecular transport through cellularmembranes via ion channels and, in particular, to a substrate providinga set of nano to microscale pores that may be readily sealed to cellularmembranes. Ion channel investigation using patch clamps often plays animportant role in drug discovery and preliminary drug screening orevaluation, for example, by providing a model that shows an effect of adrug on ion channels. Doing so can be useful for either avoiding adverseeffects or for creating a positive therapeutic effect for the treatmentof ion channel related diseases.

Drug screening can require a large number of ion channel measurements.Accordingly, in current practice, planar patch clamps are preferablebecause they allow parallelization of multiple samples on a substrate,often referred to as a wafer, chip, or well-plate, and facilitatemeasurement automation. Each sample has a cell or cell wall that ispositioned so that an ion channel in the cell or cell wall is alignedwith a pore at the sample site. The cell or cell wall is sealed to thepatch clamp substrate in a manner that allows a small amount ofelectrical current to be used in performing ion channel investigations,typically by way of an extremely high resistance seal between the patchclamp substrate and the cell wall (a gigaohm seal or gigaseal). Gigaohmseals achieved using on-chip patch clamp procedures usually haveelectrical resistance values of about 1 gigaohm, with resistance valuesof up to about 5 gigaohms being achieved in some relatively rareinstances.

Planar patch clamp substrates can be made from, for example, silicon,Teflon®, PDMS (polydimethylsiloxane), PSG (phosphosilicate glass), orglass. While such materials prove suitable for many planar patch clampimplementations, a single crystal quartz (quartz) material can be aparticularly desirable for making planar patch clamp substrates. Quartzexhibits particularly high electrical insulating properties and ispiezoelectric. Its unique electrical characteristics allow it to be usedas a patch clamp substrate by providing very low levels of backgroundnoise while performing ion channel investigations. Furthermore, quartzexhibits particularly good mechanical characteristics such as, forexample, good hardness, thermal stability, and chemical stabilitycharacteristics. Despite a general recognition of quartz's suitabilityfor use as a patch clamp substrate, many of its desirablecharacteristics, such as hardness, make fabricating (micromachining) thepores in a quartz substrate rather difficult and/or time consuming.

Traditionally, micromachining of quartz is performed using a combinationof lithography and reactive ion etching (RIE). However, RIE techniquesrequire multiple steps and are relatively slow processes.

Another method of micromachining quartz is by way of direct laser beamablation. During direct laser beam ablation, a high power density, shortpulse width femtosecond laser beam is irradiated directly onto quartz.The nonlinear interaction between the ultrafast laser pulses and quartz,which has a band gap of about 9 eV, results in a cyclic multiphotonabsorption and electron excitation between the ground and excitedstates. During this process, the initial excited electrons induce anavalanche ionization and generate a microplasma which ablates thequartz. However, since quartz has a wide band gap, this approach is alsoslow and is limited in terms of pore diameter and material thicknessthat can be achieved.

Recently, numerous advances have been made in micromachining of pores innon-quartz substrates, for example, by utilizing nanosecond lasers, suchas excimer lasers instead of femtosecond lasers. Excimer lasers, whichemit ultraviolet (UV) light, have been successfully implemented inrelatively fast drilling procedures in non-quartz materials. However,quartz has excellent optical transmission over a large spectrum, from UVto infrared (IR), whereby it is transparent to light(s) in thisspectrum. Since quartz is transparent to and therefore substantiallyunaffected by UV light(s), it has been widely accepted that excimerlasers are not usable for micromachining quartz.

Furthermore, although various patch clamping and other techniques havebeen developed and, at least to some extent, standardized forsuccessfully modeling and investigating ion channel functionvoltage-sensitive (or voltage-gated) ion channels, in-depthinvestigation of yet other types of ion channels, such asmechanosensitive ion channels, remains at least somewhat frustratingand/or impracticable. Accordingly, numerous molecular mechanisms andtheir functionalities within mechanosensitive ion channels remainunknown, whereby cellular responses to mechanical stimuli remain some ofthe least understood of the known sensory mechanisms.

SUMMARY OF THE INVENTION

The present invention provides an improved technique for the generationof nanoscale-sized pores, for example, pores having diameters of nearly200 nm, using a laser through a substrate that is substantiallytransparent to the laser's emitted wavelength and therefore tends to beunaffected by the laser using previously known practices. In thetechnique, the substrate is backed by an energy absorbing material thathas relatively high coefficients of thermal expansion and UV absorption.The laser light is transmitted through the substrate and focusedadjacent a back side of the substrate, for example, at or immediately infront of or behind an interface defined between the substrate andenergy-absorbing material. Doing so can increase a temperature of theenergy-absorbing material which, in turn, heat the substrate from beyondits back side. Heating the energy-absorbing material in this mannerincreases the temperature of the substrate to an extent that melts thesubstrate, and correspondingly facilitates melting-type formation ofsmall diameter smooth pores and can also lead to formation of acrater(s) at the back side of the substrate. According to some aspectsof the invention, the crater can be formed by a shock wave that resultsfrom thermal expansion of the energy absorbing material, but in anyevent, is formed by ablation or other thermal related removal ofmaterial from the back side of the substrate. The pore and/or crater aretherefore created by a sandwich drilling or sandwich drilling-liketechnique that can give the pore and/or crater, of the quartz chip,surface characteristics that are similar to surface characteristics ofpipettes that have been fire-polished with open flame procedures. Inother words, the pore(s) and/or crater(s) formed according to aspects ofthe present invention have surfaces that are substantially smooth, theirsurface irregularities having been removed or attenuated by surfacetension induced flowing of melted material in such irregularities whichtends to smooth the same.

Specifically then, the present invention provides a method of creatingnanoscale holes by using steps including creating a multi-layeredassembly that comprises a substrate material and an energy absorbingmaterial. The substrate material receives the nanoscale hole and definesa front side and an opposing back side. The energy absorbing material isadjacent the back side of the substrate material. A laser is appliedthrough the multi-layered assembly, by initially passing through thesubstrate material and being focused at and absorbed by the energyabsorbing material, increasing a temperature of the back side of thesubstrate material to a greater extent than an increase in temperatureof the front side of the substrate material. Continued application ofsuch laser stimulus to the energy absorbing material may correspondinglyheat and/or pressurize the back side of the substrate, which caninitiate a drilling of or establishing a pore through the substrate.

It is therefore an object of at least one embodiment of the invention toutilize a laser to drill a pore in a substrate by indirectly heating thesubstrate by focusing the laser at or near an interface between thesubstrate and an energy absorbing material. This allows for lasermicromachining of a material that might otherwise be transparent tolight of a wavelength emitted by the laser.

The method can further include a step of producing a crater at the backside of the substrate material which, in some embodiments, can beproduced by a shock wave that increases both a temperature and apressure at an interface defined between the back side of the substratematerial and the energy absorbing material. The crater formation can bean initiating step for drilling of the pore and the pore, in someembodiments, is drilled in a drilling direction that opposes a directionof laser transmission through the substrate.

It is thus an object of at least one embodiment of the invention toprovide a method for forming craters in back sides of substrates and/ordrilling pores from the crater toward the front side of the substrate.

The substrate material may be a single crystal quartz material and thelaser can be a UV emitting excimer laser or other lasers, e.g., CO2lasers, and/or others.

It is therefore an object of at least one embodiment of the invention togenerate pores and/or craters in a single crystal quartz wafer or chipand may be a further object of at least one embodiment of the inventionto perform laser micromachining on a substrate that is substantiallytransparent to a wavelength of light that is emitted by the laser.

The energy absorbing material may be a liquid media, for example, anultraviolet radiation absorbing organic liquid such as acetone and/orfluorescence immersion oil. Such liquid media can have a thermalexpansion coefficient of at least about 700×10−6 K⁻¹ and an ultravioletabsorption coefficient of at least about 1.46 cm⁻¹, for example, anultraviolet absorption coefficient of between about 1.46 cm⁻¹ to 1.65cm⁻¹.

It is thus an object of at least one embodiment of the invention toprovide a liquid medium for use as an energy absorbing material that canbe placed under the substrate for performing a laser induce backsideetching of the back side of the substrate. The backside etching may beperformed on a quartz chip that has been pre-etched or thinned from aninitially thick substrate. The initially thick substrate may be about100-500 microns thick and can be either laser or wet etched, forexample, with a buffered oxide etchant (BOE) or hydrofluoric (HF)etchant, in a pre-thinning procedure, down to about 20-50 microns inthickness before the nanopore laser drilling is initiated, which mayimprove suction and reduce series resistance.

The pore can have a smaller diameter than the crater which itintersects, and the pore diameter can be generally constant along itslength or a major portion thereof, and a crater sidewall can include anundercut or groove extending thereinto.

It is thus an object of at least one embodiment of the invention toprovide a patch clamp chip that has a smooth pore that opens into arelatively larger diameter crater. This configuration can providesuitable structure to which a cell can be mounted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an apparatus used for producing aplanar patch clamp chip or wafer per from a multi-layered assembly oneembodiment of the present invention;

FIG. 2 is a pictorial view of the multi-layered assembly used in themachine of FIG. 1;

FIG. 3 is a cross-section along line 3-3 of FIG. 2 showing a spacing ofa substrate from a backer plate by a gap filled with an energy absorbingmaterial;

FIG. 4 is a figure similar to that of FIG. 3 showing an initial stage oflaser micromachining in which a laser beam is focused toward aninterface between the substrate and energy absorbing material;

FIG. 5 is a figure similar to that of FIG. 3 showing transmission ofenergy through the substrate into the energy absorbing material after acrater has been formed in a back side of the substrate;

FIG. 6 is a figure similar to that of FIG. 5 showing an initiation of apore drilling procedure;

FIG. 7 is a figure similar to that of FIG. 5 showing the pore aftercompletion of the drilling procedure;

FIG. 8 is an enlarged scanning electron microscope image of a substrateafter focused ion beam milling to show a cross-section of a firstembodiment of a substrate that was laser micromachined according tomethods of the invention;

FIG. 9 is an enlarged scanning electron microscope image of a pictorialview of a variant of the substrate of FIG. 8;

FIG. 10 is an enlarged scanning electron microscope image of a pictorialview of another variant of the substrate of FIG. 8;

FIG. 11 is an enlarged scanning electron microscope image of a pictorialview of a second embodiment of a substrate that was laser micromachinedaccording to methods of the invention;

FIG. 12 is a simplified representation of the use of the substrate ofFIG. 7 in a patch clamp application; and

FIG. 13 is a simplified representation of another use of the substrateof FIG. 7 in a patch clamp application that incorporates piezoelectricactuation controls.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Referring now to FIG. 1, the present invention may use an ArF excimerlaser 10 having collimating and focusing optics 11 to direct a narrowcollimated beam 12 of light along an axis 15 toward a back surface of asandwich-like, multi-layered assembly 14 that is held on a mechanicalstage 16. The laser may, for example, have a wavelength range of about155 nm to about 195 nm and is preferably operated to emit a beam 12 oflight having a wavelength of about 193 nm. In other embodiments, laser10 may be yet other types of lasers, such as CO2 lasers, and/or others,depending on, for example, a desired wavelength that is selected basedon particular characteristics of components within the multi-layeredassembly 14, in other words, how the multi-layered assembly 14 reacts toexposure to light having such wavelength(s), and/or other factors.

The laser 10 may include a variable attenuating mirror for controllinghow much of the laser beam is to be transmitted, and a stencil metalmask with an adjustable aperture that allows production of differentlaser beam shapes and sizes. Laser 10 may further include a stage 16that can be controlled by an automated controller 18 of the type wellknown in the art for providing control signals 22 to the laser 10,controlling its output power in a series of pulses as will be describedand providing control signals 22 to actuator motors 24 providing x-ycontrol of the stage 16.

Referring now to FIGS. 2 and 3, the multi-layered assembly 14 mayinclude a substrate 26, an underlying energy absorbing material 34, anda backer plate 32 that supports the energy absorbing material 34 andsubstrate 26. The energy absorbing material 34 cooperates with thesubstrate 26 to perform a one-step micromachining process in which thelaser 10 drills through the substrate 26, forming small diameter smoothholes therethrough, while causing a nominal amount of surface roughnessto the substrate 26. For example, a synergistic relationship between theenergy absorbing material 34 and the substrate 26 allow for using thelaser 10 to perform a one-step micromachining process to create holes orpores that have diameters as small as 200 nm, while resulting in anend-of-procedure surface roughness of merely tens of nanometers asmeasured from the respective surface(s) of the substrate 26.Furthermore, submicrometer holes, bores, or pores may be formed in thesubstrate 26 along with crater-shaped depressions that are formed in asurface of the substrate that faces a direction of back from thesubstrate. Such relationships between the components of multi-layeredassembly 14, and techniques for forming such hole, pores, and craters inthe one-step micromachining procedure, are discussed in greater detailelsewhere herein.

Still referring to FIGS. 2 and 3, within the multi-layered assembly 14,substrate 26 can be, for example, a clear solid material that istransparent to the UV light emitted by laser 10 (FIG. 1), preferably asingle crystal quartz chip or wafer. A suitable such single crystalquartz wafer for use as substrate 26 is the 350 μm thick quartz waferthat is readily available from the University Wafer Company of SouthBoston, Mass.

A front surface of the upper substrate 26 may have a series ofdepressions or wells 28 formed at regular x-y grid locations 29. Thewells 28 provide a thinned portion 30 at the locations 29 measured alongaxis 15 having a thickness of 100 to 1000 g and may be molded, ground,or etched in the substrate 26. The diameter of the wells 28 may berelatively large, for example, 5.0 mm, and serve simply to permit agenerally thicker substrate 26 in regions outside of the locations 29for structural convenience. Referring specifically to FIG. 2, thesubstrate 26 can define a single, unitary structure with multiple wells28 formed therein. However, it is also contemplated that substrate 26can be an assembly of multiple, relatively smaller, individual quartzwafers or chips, each having only a single well 28 formed therein.

Referring yet further to FIGS. 2 and 3, in some embodiments, backerplate 32 is positioned as a lowermost component of the multi-layeredassembly 14 and can serve as, for example, a structural base or part ofa structural frame thereto. Backer plate 32 may be a supportive slidethat is incorporated into the multi-layered assembly 14, underlying andbeing spaced vertically below the substrate 26. Optional spacer 31 isone suitable structure that can be used to establish such verticalspacing between the substrate 26 and backer plate 32. The spacer 31 canbe formed out of, for example, polydimethylsiloxane (PDMS). The PDMS maybe cast on the rear surface of the substrate 26 through a mold producedusing integrated circuit techniques to provide precisely controlledspacer thickness or may be spun-coated and selectively removed except atthe edges of the substrate 26. Regardless of the particular manner inwhich the PDMS is incorporated into the multi-layered assembly 14, it ispreferably configured to form a chamber defined by the PDMS at its outerperimeter and defining a space between the substrate 26 and backer plate32.

The space between the substrate 26 and the backer plate 32 is filledwith energy absorbing material 34 which can be a liquid media and, insome embodiments, a UV absorbing organic liquid. The energy absorbingmaterial 34 has thermal expansion and/or UV absorption coefficients thatare large enough to heat and/or squeeze the substrate 26 to an extentthat facilitates, catalyzes, or initiates laser drilling of thesubstrate 26. The energy absorbing material 34 can have a thermalexpansion coefficient of at least about 700×10⁻⁶K⁻¹ and a UV absorptioncoefficient of at least about 1.46 cm⁻¹, preferably being within a rangeof between about 1.46 cm⁻¹ to 1.65 cm⁻¹. Suitable UV absorbing organicliquids for use as energy absorbing material 34 include, for example,acetone and fluorescence immersion oil, along with other suitablematerials that may increase temperature when exposed to UV radiation toan extent that may correspondingly heat the substrate material,interfacing therewith, so as to melt the substrate material. Examples ofother suitable energy absorbing materials 34 include, but are notlimited to, pyrene, naphthalene, and toluene, and/or other materials.

Still referring to FIGS. 2 and 3, the particular amount of volume ofenergy absorbing material 34 is selected based on the intendedconfiguration of multi-layered assembly 14, and laser drillingtechniques that are implemented, and intended end-use configuration andcharacteristics of substrate 26 for its use in a patch clamping setup.In some embodiments, a relatively small amount of energy absorbingmaterial 34 is used by sandwiching a thin layer of the energy absorbingmaterial 34 between backer plate 32 and substrate 26, withoutincorporating spacers 31. In other embodiments, such as the oneillustrated in FIG. 2, a relatively greater volume of energy absorbingmaterial 34 is provided, with the particular volume being a function ofthe space between the backer plate 32 and substrate 26 as dictated byspacer 31.

Regardless of whether the energy absorbing material 34 is implemented asa thin layer that is tightly sandwiched between the substrate 26 and abacker plate 32 substantially adjacent and below the substrate 26, orimplemented as a thicker layer that fills a larger space between thesubstrate 26 and backer plate 32 as dictated by spacer 31, the energyabsorbing material 34 and substrate 26 are preferably in a face-to-faceor an abutting relationship with respect to each other. In suchconfiguration, the substrate 26 and energy absorbing material 34 definean interface 35 therebetween. Interface 35 allows the energy absorbingmaterial 34 to transmit heat and/or pressure to the substrate 26 withrelatively little energy loss in so doing.

Referring now to FIGS. 1 and 4, the laser 10 may be positioned above afirst location 29 and pulsed by the controller 18 to produce a series ofcontrolled light pulses 40 of laser beam 12 that is focused at theinterface 35 or substantially adjacent or proximate thereto. The lightpulses 40 are directed toward a front side 46, passing through an outersurface 47 thereof. Since substrate 26 is substantially transparent toUV light, the light pulses 40 pass through the entire thickness of thesubstrate 26, leaving the substrate 26 through an outer surface 49 of aback side 48 of the substrate 26. At this point, the substrate 26 issubstantially unheated or otherwise affected by the light pulses 40, atleast compared to the temperature increase, thermal expansion, and/orother responses of the energy absorbing material to the stimulus of thelight pulses 40.

Still referring to FIGS. 1 and 4, the laser 10 indirectly heats thesubstrate 26 by primarily heating the energy absorbing material 34which, in turn, secondarily heats the substrate 26 at its back side 48or from below. In particular, the light pulses 40 that are focused atthe interface 35, heat the energy absorbing material 34 and thereforealso the outer surface 49 of the back side 48 of substrate 26 toestablish rapid increases temperature and pressure at the interface 35.This correspondingly leads to rapid thermal expansion of the energyabsorbing material 34 and/or exit side 48 of substrate 26 that can leadto an ablation of material from the exit side 48 or, in someembodiments, create a shock wave 52 at the interface 35. In any event, aone-step emission of the laser 10 may be used for wholly fabricating acrater 53 and/or pore 54 in the substrate 26, explained in greaterdetail elsewhere herein. The particular configuration andcharacteristics of the crater 53 and/or pore 54 are influenced by,amongst other things, the particular setup of the laser 10 and itsoutput qualities, described in greater detail elsewhere herein, wherebythe craters 53 and/or pores 54 are give desired sizes, shapes, and/orother characteristics by selecting a corresponding setup and outputqualities of the laser 10.

Referring now to FIGS. 5-8, shows an embodiment of crater 53 that can bea disc-shaped depression extending into the back side 48 of thesubstrate. Crater 53 has a relatively flat bottom wall 102 that can havea circular perimeter shape and extend generally parallel to the outersurface 49 of back side 48. Seen best in the SEM (scanning electronmicroscope) image of FIG. 8, crater 53 of this embodiment includes asidewall 105 that may taper slightly inwardly toward the bottom wall102. An upper portion of sidewall 105, in its orientation of FIG. 8,transitions into the outer surface 49 of back side 58 by way of anarcuate surface that may have been at least partially smoothed duringits formation. The smoothing is analogous to the smoothing achieved onan exponentially larger scale with open flame fire polishing, forexample, while fire polishing a pipette or some other structure that isexponentially larger than the crater 53 and/or pore 54.

Intuitively, overall dimensions of the crater 53 are functions ofdimensions and characteristics of the sidewall 105. Stated another way,crater 53 defines a crater depth which corresponds to a height dimensionof the sidewall 105. A crater width or diameter is defined by a(greatest) distance measured between facing surfaces of the sidewall105. As can be extrapolated from the size scale provided in FIG. 8,crater 53 may have a crater diameter of about 40 μm and a crater depthof about 10 μm.

In some embodiments, along at least part of the perimeter of crater 53,an undercut 110 extends radially into the sidewall 53, between thesidewall and the bottom wall 102. Such undercut 110 may define a grooveas an interlocking structure into which portions of a cell can flowunder certain conditions, allowing parts of the cell's membrane toengage against, for example, a projecting shoulder defined between theundercut 110 and sidewall 105.

Referring now to FIGS. 7 and 8, after completion of the lasermicromachining of substrate 26, a smoothed annular edge or shoulder candefine an opening between the crater 53 and a pore 54, such openingbeing labeled as a pore-crater opening 153. Like various portions of thecrater 53, the pore-crater opening 153 can be fire polished and smoothedso as to eliminate substantially all surface irregularities. Seen bestin FIG. 8, the pore-crater opening 153 maybe located in the middle ofbottom wall 102 of the crater.

The pore 54 extends between the pore-crater opening 153 and apore-surface opening 147 that opens into the pore 54 from the outersurface 47 of the front side 46 of substrate 26. Accordingly, inembodiments of substrate 26 that include a crater 53 formed thereinto,pore 54 extends between the crater 53 and the front side 46. Inembodiments of substrate that do not include a crater 53, the pore 54extends between the front and exit sides 46, 48, in other words, throughthe entire thickness dimension of the substrate 26. Regardless of theparticular end-use configuration of substrate 26, pore 54 has asubstantially constant diameter along at least a major portion of itslength. Accordingly, the pore-surface and pore-crater openings 147, 153may define opening diameters that are about the same size, for examplewith the larger of the two openings being no more than about 50% greaterthan the smaller of the two openings. As another example, the pore 54can define minimum diameter and maximum diameter segments along itslength, with the maximum diameter segment being no more than about 50%greater in magnitude than the smaller diameter segment.

Referring to FIGS. 4-8, a crater 53 and/or pore 54 may be formed intosubstrate 26 of the multi-layered stack 14 in the following way. Themulti-layered stack 14 is assembled and the laser 10 is set up to basedon intended characteristics of the crater 53 and/or 54, such as porediameter and/or others. As discussed above, light pulses 40 are focusedaway from the front side 46 of the substrate 26 and toward its interface35 with the energy absorbing material 34. Doing so causes a thermalexpansion and also pressure increase of the affected material(s) withinthe multi-layered stack 14, which may be largely the energy absorbingmaterial 34 at this early stage. Correspondingly, the interface 35 andthe back side 48 of the substrate 26 may be secondarily affected by thechanges occurring within the energy absorbing material 34. Suchsecondary affects may be an indirect heating characteristic of laser 10,by way of the substrates' 26 intimate interaction with the energyabsorbing material 34.

For example, by focusing the light pulses proximate the interface 35,increasing temperature and pressure of the energy absorbing material 34can be transmitted to the substrate 26, establishing a localized zone ofincreasing temperature and pressure of the back side 48 nearest thepoint of focus of the light pulses 40. This may cause temperature andpressure differentials between the front and exit sides 46, 48 of thesubstrate 26 but in any event will increase temperature and pressure atthe back side 48. When such values increase enough, a crater 53 and/orpore 54 can be established by way of this a one-step micromachiningprocedure.

Referring now to FIGS. 5-7, although the Applicant does not wish to bebound by a particular theory, it is contemplated that the formation ofcrater 53 may be an initiator of the drilling of pore 54. In suchembodiments, once crater 53 is formed, the near molten material ofbottom wall 102 is more receptive to accepting energy from or, in otherwords, is less transparent to the UV from laser 10 than material in aroom temperature at-rest substrate 26. Accordingly, due to thepreheating of bottom wall 102 during establishment of the crater 53, thelight pulses 40 are able to further ablate material at their point ofinteraction with the preheated bottom wall 102, allowing the lightpulses 40 to pierce therethrough and begin formation of the columnarpore 54.

Referring now to FIGS. 6 and 7, while the emission of light pulses 40continues, so does the ablation or melting away of more material at theparticular location which the light pulses 40 pass through the materialof the substrate (FIG. 6). In this regard, the pore 54 may propagateupwardly toward the front side 46 of the substrate so as to define adrilling direction “D” that opposes the direction of the light pulses 40passing through the substrate 26. In yet other embodiments, the pore 54is not formed in the drilling direction “D” but instead, the drillingdirection can extend in the same direction as the light pulses 40passing through the substrate 26, whereby the pore 54 drilling mayoriginate at the outer surface 47 of the front side 46. Although theterms “front” and “back” in describing various portions of the substrate26 have been implemented in a convenient sense, it is fully contemplatedthat the by, for example, inverting the arrangement of the components ofstack 14, or positioning the laser 10 on the opposing side of the stack14, such terms might then assume generally opposite means. In otherwords, regardless of the particular orientation of the stack 14 and/orrelative positions between the stack 14 and laser 10, in preferredembodiments, the drilling direction “D” extends from the interface 35between the energy absorbing and substrate materials 34, 26, toward theside or outer surface of the substrate 26 that opposes the energyabsorbing material. That is, the drilling direction “D” typicallyextends away from the location where the shock wave 52 occurred, whethersuch direction is the same as or opposite to the direction of laser 10emission.

Regardless of how the pore 54 is created in a particular embodiment, thepore characteristics such as, for example, pore diameter, may becontrolled or manipulated at least to some extent by adjusting the setup and controls of the laser 10. For some crater 53 and/or pore 54formation procedures, the laser output power may be fixed at 5 W and avariable attenuator of the laser 10 can be set to allow about 70%, forexample, 73% of total beam transmission, and operated for 2500 pulses ata 100 Hz repetition rate for the light pulses 40. Yet other set ups arecontemplated, again, based on the intended characteristics of the crater53 and/or pore 54 that are being formed.

For example, Table 1 below illustrates how pore diameter can beinfluenced by changing transmission rate or repetition rate of theemitted light pulses 40, from 50 Hz pulses to 100 Hz pulses during theiremission from the laser 10. To establish such data of Table 1, laserenergy was fixed at 50 mJ and six drilling schemes (Recipes) were testedto show various combinations of the two transmission rates, and thecorresponding influence on pore diameter.

TABLE I Drilling Recipes for Two Different Transmission Rates and TheirResults Pore Diameter Pore Diameter 50 Hz 100 Hz (μm) at 83% (μm) at 87%Recipe Pulses Pulses Transmission Rate Transmission Rate 1 3000 00.216^(a) 3.841 2 500 2000 0.621 4.168 3 1000 4000 0.342 4.455 4 15003000 0.395 4.511 5 2000 2500 1.594 5.270 6 2000 4000 3.210 5.421^(a)Note: This approaches the laser wavelength limit of 193 nm.

Referring generally to FIGS. 8-11, the different configurations of thesedifferent embodiments show that, similar to the data in Table 1,influencing output characteristics of laser 10 may be used to formcraters 53 and/or pores 54 that have different features, configurations,and/or other characteristics, as desired. Namely, using (relatively)higher transmission rates for laser 10, for example, transmission ratesof greater than about 80%, produces craters 53 with flat bottom walls102, like those seen in FIGS. 8-10. Using (relatively) lowertransmission rates for laser 10, for example, transmission rates thatare less than about 80%, produces craters 53 that have taperingsidewalls and substantially no discernable bottom wall, like that seenin FIG. 11.

Specifically regarding the embodiments of FIGS. 8-10, of the embodimentsof craters 53 that have flat bottom walls 102 or semisphericalconfigurations, crater depth and sidewall configuration can also becontrolled by the transmission rate or power of laser 10. Of the threedifferent embodiments shown in FIGS. 8-10, the embodiment of crater 53of FIG. 10 has the most shallow crater depth and also has a semicircularor concave-up, arcuate transition between the bottom wall 102 and thesidewall 105. The crater 53 of FIG. 10 that was formed with atransmission rate of laser 10 of about 83%.

Again comparing the embodiments of FIGS. 8-10, the crater 53 of FIG. 9has a deeper crater depth than that of FIG. 10 but is shallower thanthat of FIG. 8. The crater 53 of FIG. 9 has a similar semicircular orconcave-up, configuration to that seen in FIG. 10, with a somewhatflatter bottom wall 102 when compared thereto. The crater 53 of FIG. 9was formed with a transmission rate that is between 83% and 87% whichwas used in making craters 53 of FIGS. 10 and 8, respectively. Thecrater of FIG. 8 has been previously discussed and, when compared to thecraters 53 of FIGS. 9 and 10, has the deepest crater depth and theflattest bottom wall 102, deviating the most from a semicircularcross-sectional configuration. As discussed elsewhere in greater detail,the crater 53 of FIG. 8 includes an undercut 110 that is defined betweenthe bottom and sidewalls 102 and 105.

Referring now to FIG. 11, this embodiment of crater 53 is made bysetting the laser 10 to a relatively low transmission rate, for example,a rate of about 74%. This sub-80% transmission rate forms a crater 53that appears trumpet-like or arcuately tapering in cross-section, suchthat the sidewall 105 tapers conically down to where it connects to thepore 54. The sidewall 105 can also, in some related embodiments, havescales or patterned discontinuities across surfaces thereof.

Referring now generally to FIGS. 12-13, regardless of the particularconfiguration of crater 53 and pore 54, after the substrate 26 has beenlaser micromachined into a usable wafer or chip having a crater 53and/or pore 54, it can be implemented into a suitable investigative toolor instrument, depending on the particular intended end-use research orstudy that will be performed. It has already been shown that the craters53 of the invention can provide gigaohm seals that are not onlysatisfactory in performance, but may be notably superior when comparedto currently known techniques. For example, whereas known methods ofglass chip production yield about 60% of produced units that can achieve1.0-1.5 gigaohm seals, and a substantially lower percentage that canachieve about 5.0 gigaohm seals, preliminary mockup productions of glasschips using the inventive procedure(s) have already successfullyproduced a substantial percentage of produced units that have achieved7.0 or greater gigaohm seals. In view of such promising preliminaryresults, it is fully contemplated and expected that the inventivemethods disclosed herein are fully capable of producing chips thatachieve or approach 15 gigaohm sealing capabilities.

Referring specifically now to FIG. 12, in some embodiments, thesubstrate 26 can be used in a planar patch clamp apparatus toinvestigate ion channel performance. As one example of suchinvestigation, the substrate 26, shown having the same orientation asseen in FIG. 8 and therefore an inverted orientation with respect tothat shown in FIG. 7, may receive a cell 60 within the crater 53 toexpose a portion of the cell wall 62 to be accessible through the pore54. A light suction applied by a pump 67 from the front side 46 mayadhere the cell wall 62 to the surface of crater 53 with a 5 to 30gigaohm resistance between a solution 64 on the side of the substrate 26holding the cell 60 and a solution 66 on the side of the substrate 26opposite solution 64. The application of suction may correspondinglyalso pull a portion of the cell wall 62 into the undercut 110 in amanner that enhances the seal of the cell 60 to the substrate 26 by wayof the mechanical interlocking relationship therebetween. Although thecell 60 is shown in FIG. 12 as having its membrane or cell wall 62ruptured over the pore 54, it is, of course, contemplated that the cellwall 62 remains intact for various other types of studies orinvestigations.

A sharp suction applied by a pump 67 at the outer surface 47 of thefront side 46 or other means may be used to provide electricalconnection to the interior of the cell 60 by a sensitive electricaldetector 70 permitting measurement of electrical differences between theexterior and interior of the cell 60 through an electrode 72communicating with the interior of the cell 60 referenced to solution 64outside the cell 60.

Referring now to FIG. 13, since preferred embodiments of substrate 26are made from a single crystal quartz material, such embodiments may beincorporated into an apparatus to investigate mechanosensitive ionchannel performance and function. The apparatus of FIG. 13 is largelyanalogous to that of FIG. 12, only being configured to piezoelectricallyactuate, stress, or otherwise stimulate the cell wall 62 so as tomeasure, by way of detector 70, gating responses of the particular ionchannel that cooperates with the pore 54.

Still referring to FIG. 13, a bather 200 can be provided that acts as anenclosure, retaining the solution 64 therein. Ends of the substrate 26extend through opposing sides of the barrier 200. A power source 210provides electrical stimulus for stimulating the piezoelectric behaviorsof the substrate. A controller 220 sends and controls an electricalsignal to the substrate, through conductors 225 that lead to thesubstrate. The actual connection(s) of the conductors 225 to thesubstrate 26 can be accomplished with suitable terminals. For example,terminals 230 and 235 are attached to the opposing outer surfaces 46 and47 at a first end of the substrate 26, appearing as a left end in FIG.13, and connected to the controller 220 by a first pair of conductors225. Terminals 240 and 245 are attached to the opposing outer surfaces46 and 47 at a second end of the substrate 26, appearing as a right endin FIG. 13, and connected to the controller 220 by a second pair ofconductors 225.

Referring yet further to FIG. 13, in such an embodiment, the controller220 may place the cell wall 62 under compressive and/or tensile stressesalong multiple axes of movement or actuation. Depending on theparticular cut of the crystal, controller 220 may establish a voltageacross the thickness of the substrate 26, specifically by establishing avoltage between the upper terminals 235, 245 and the lower terminals230, 240. Depending on the polarity of the signal, doing so will causethe substrate 26 to compress or elongate with respect to its thicknessdimension which correspondingly compresses or stretches the cell wall 62in such direction. Here too, depending on the particular cut of thecrystal, controller 220 may establish a voltage across the length of thesubstrate 26, specifically by establishing a voltage between the leftend terminals 230, 235 and the right end terminals 240, 245. Againdepending on the polarity of the signal, doing so will cause thesubstrate 26 to compress or elongate, only this time with respect to itslength dimension, compressing or stretching the cell wall 62 in acorresponding manner. While doing so, the detector 70, senses notablegating and/or other responses of the particular ion channel or otherportions of the cell 60, depending on the particular configuration ofdetector 70.

Referring yet further to FIG. 13, regardless of the particularconfiguration of the cell membrane investigating apparatus and its setupand controls, the substrate 26 according to the invention provides an“on-chip” piezoelectric system that may be used as a suitablealternative for imposing mechanical deformations to a membrane which todate has been primarily studied with pipettes and conventional patchclamping. Substrate 26 can be configured and implemented specificallyfor ones of, e.g., (i) lower frequency actuation of the ion channels,(ii) higher frequency mechanical probing, as well as (iii) static stressmodulation of the membrane, as desired based on the particularinvestigation being performed.

It is specifically intended that the present invention not be limited tothe embodiments and illustrations contained herein, but include modifiedforms of those embodiments including portions of the embodiments andcombinations of elements of different embodiments as come within thescope of the following claims.

1. A method of creating nanoscale holes comprising the steps of: (a)creating a multi-layered assembly comprising, (i) a substrate materialreceiving a nanoscale hole therethrough and having opposing outersurfaces defining a thickness therebetween; and (ii) an energy absorbingmaterial being adjacent one of the opposing outer surfaces so as todefine an interface between the substrate and energy absorbingmaterials; (b) after creating the multi-layered assembly, applying alaser through the multi-layered assembly so that is passes into and hasenergy absorbed by the energy absorbing material; (c) producing a shockwave at the interface to remove material from and create a hole throughthe entire thickness of the substrate material in a direction ofpropagation that begins at the interface and extends toward the outersurface of the substrate material that opposes the interface.
 2. Themethod of claim 1, further comprising a step of producing a crater at aback side of the substrate material.
 3. The method of claim 2 whereinthe shock wave increases both a temperature and a pressure at theinterface.
 4. The method of claim 2 wherein producing the craterinitiates the forming of a pore in a drilling direction that opposes adirection of laser transmission through the substrate.
 5. The method ofclaim 4 wherein the substrate material is a single crystal quartzmaterial and the energy absorbing material is a liquid media.
 6. Themethod of claim 5 wherein the liquid media comprises an ultravioletradiation absorbing organic liquid.
 7. The method of claim 6 wherein theultraviolet radiation absorbing organic liquid comprises at least one ofacetone and fluorescence immersion oil.
 8. The method of claim 5 whereinthe liquid media has a thermal expansion coefficient having a magnitudethat allows the liquid media to heat the single crystal quartz materialto the melting point of the single crystal quartz material, when thelaser is applied through the multi-layered assembly.
 9. The method ofclaim 5 wherein the liquid media has an ultraviolet absorptioncoefficient of at least about 1.46 cm⁻¹.
 10. The method of claim 9wherein the liquid media has an ultraviolet absorption coefficient ofbetween about 1.46 cm⁻¹ to 1.65 cm⁻¹.
 11. A method of creating nanoscaleholes comprising the steps of: (a) creating a multi-layered assemblycomprising, (i) a substrate material receiving the nanoscale hole; and(ii) an energy absorbing material being adjacent an outer surface of thesubstrate material; (b) applying a laser through the multi-layeredassembly; (c) focusing the laser at a position within the multi-layeredassembly that is proximate the energy absorbing material and absorbingfrom the laser with the energy absorbing material; (d) heating an outersurface of the substrate material that is closest to the energyabsorbing material to a greater extent than an outer surface of thesubstrate material that is furthest from the energy absorbing material;(e) producing a crater in the outer surface of the substrate materialthat is closest to the energy absorbing material.
 12. The method ofclaim 11, wherein the substrate material defines a front side and a backside, and wherein the energy absorbing material is adjacent the backside of the substrate material such that the crater extends into theback side of the substrate material.
 13. The method of claim 12, furthercomprising generating a pore that extends continuously between thecrater and an outer surface of the front side of the substrate material.14. A patch clamp chip for electrophysiology comprising: a substratehaving a first outer surface and an opposing second outer surface; acrater extending into one of the first and second outer surfaces anddefining a crater diameter thereon; and a pore extending between thecrater and the other one of the first and second outer surfaces of thesubstrate, the pore defining a pore diameter that is smaller than thecrater diameter and is generally constant along a major portion of alength of the pore.
 15. The patch clamp chip of claim 14 wherein thesubstrate material is a single crystal quartz material that has beenpre-thinned by at least one of a laser etching and a wet etchingprocedure.
 16. The patch clamp chip of claim 14, the pore defining (i) apore-crater opening diameter defined at a location where the pore opensinto the crater, and (ii) a pore-surface opening diameter defined at alocation where the pore opens through an outer surface of the substratematerial, and wherein the larger of the pore-crater and pore-surfaceopening diameters is no more than about 50% greater than the smaller ofthe pore-crater and pore-surface opening diameters.
 17. The patch clampchip of claim 14, the pore defining a minimum diameter segment along alength thereof and a maximum diameter segment along the length thereofand wherein a diameter of the maximum diameter segment is no more thanabout 50% greater than a diameter of the minimum diameter segment. 18.The patch clamp chip of claim 14, further comprising a shoulder definedbetween the pore and a generally flat bottom wall of the crater suchthat a longitudinal traverse between the crater and the pore defines astep-change between respective diameters of the crater and pore.
 19. Thepatch clamp chip of claim 18, the crater including (i) a crater depthbeing defined between a crater opening and the crater bottom wall, and(ii) a crater width being defined transversely across the crater bottomwall, and wherein crater width is at least two times greater than thecrater depth in magnitude.
 20. The patch clamp chip of claim 14, thecrater including (i) a bottom wall, (ii) a sidewall extending outwardlyaway from the bottom wall, and (iii) an undercut that extends into thesidewall so as to define a sidewall shoulder between the undercut andthe sidewall.